for microarray analysis yielded RNA integrities greater than 8. 0 using an Agilent Bioanalyzer. Microarray analysis employed Affymetrix rat genome 230 2 arrays and was performed using the recommended procedures of the manufac turer. Microarray data has been deposited in NIH GEO. Data filtering and mining Differentially expressed genes were identified using BRB Array Tools selleckchem version 3. 6. 2, developed by Dr. Richard Simon and Amy Peng Lam. Data from the Affymetrix plate reader was loaded directly into the software. Affy metrix Present Absent calls were not included in the analysis. Predefined BRB Array Tools software settings were used for normalization and filtering. Data for each array were normalized using the median for the entire array. Expression values were set to 10 when they were below this value.
Expression values were excluded unless the values for at least 20% of the arrays were 1. Inhibitors,Modulators,Libraries 5 fold or more different from the median for that probe set. The significance of differences Inhibitors,Modulators,Libraries in expression among groups was determined using an F test, with significance set at p 0. 05. Because the number of genes modulated by nandrolone at each time point was not very large, all probes yielding a significant difference at p 0. 05 were included in subsequent analysis. For the comparison of gene expression in denervated muscle at 7 and 35 days, a much larger number of genes was identified, to limit the list of candidates somewhat, only those differing at p 0. 01 were included in subsequent analysis. ehicle at 7 or 35 days were calculated using geometric means.
Biological functions of differentially expressed genes were determined using Ingenuity Pathways, NIH DAVID and GeneCards at. Subsets of genes Inhibitors,Modulators,Libraries regulated by nandrolone at 7 or 35 days were selected for additional analysis based upon known or proposed relationships to muscle atro phy and hypertrophy, or transcriptional Inhibitors,Modulators,Libraries regulation by androgens. Heat maps were generated using the microarray expression data that had been normalized relative to the mean for all expression values for the array and were generated using TM4 MultiExperiment Viewer Version 4. 3. 02 . Fold change for the expres sion value for each gene and microarray was calculated relative to the arithmetic mean for the vehicle group for that gene. Tests for enrichment of biological themes were per formed using Ingenuity Pathways Analysis.
Carfilzomib Quantitative real time PCR Total RNA was used to prepare cDNA libraries by reverse transcription. Real time PCR was performed in triplicate, and the mean for protocol the crossing points of triplicates was used in subsequent cal culations. Data were normalized relative to 18S RNA. Levels of gene expression were expressed as fold change relative to denervated muscle from animals that were administered vehicle and sacrificed at 7 days using the 2 Ct method. Data are shown as mean SEM. Western blotting Gastrocnemius was homogenized in 200 ul of ice cold lysis buffer containing protease and phosphatase inhibitors using a Polyt